Thromboembolic disorders, including both venous and arterial thrombosis, are a major cause of morbidity and mortality worldwide. They are caused by dysregulation of normal blood coagulation (hemostasis) leading to abnormal formation of clots (thrombi) from fibrin, eventually resulting in tissue ischemia and, in some circumstances, embolization due to dislodging and migration of clot fragments from the thrombus.
Under normal circumstances, hemostasis is a vital mechanism that prevents blood loss from sites of vascular injury by inducing platelet activation and formation of fibrin. On a mechanistic level, hemostasis proceeds in two steps. During primary hemostasis platelets adhere to the site of trauma and become activated, and ultimately aggregate by binding to each other to form a platelet plug. Platelet plug formation is enhanced and stabilized during secondary hemostasis—a series of enzymatic reactions involving coagulation proteins (also called blood coagulation system) that culminate in formation of the protease thrombin, which converts fibrinogen to fibrin to form a stable clot that seals a breach in blood vessel walls.
Since 1964, when Macfarlane (Nature. 1964 May 2; 202:498-9) introduced the cascade hypotheses for the process of blood coagulation, the knowledge of the function of blood coagulation in vivo has grown. In the last years, the theory of two distinct routes, the so called the extrinsic and intrinsic pathway, that initiate coagulation and converge in a common pathway, ultimately leading to thrombin generation and fibrin deposition, has been revised.
In the current model initiation of coagulation occurs when the plasma protease activated factor VII comes into contact and thereby forms a complex with Tissue Factor (TF). This Tissue Factor-FVIIa complex converts the zymogen FX to its active form FXa, which in turn cleaves prothrombin (coagulation factor II) to form thrombin (IIa) in the presence of the cofactor FVa. Thrombin, a key player in coagulation, in turn can catalyze the conversion of fibrinogen into fibrin. Additionally, thrombin activates specific receptors expressed by platelets, which leads to the activation of the latter. Activated platelets in combination with fibrin are essential for clot formation and therefore are fundamental players of normal hemostasis. The FVIIa-TF complex also converts FIX to the protease FIXa, which, in the presence of FVIIIa, activates additional FX to sustain thrombin production.
The coagulation pathway involves the coagulation factor XI (FXI). It is well confirmed that FXI is, like the other members of the coagulation cascade, a plasma serine protease zymogen with a key role in bridging the initiation phase and the amplification phase of blood coagulation in vivo (Davie E W et al., Biochemistry. 1991 Oct. 29; 30(43):10363-70, Gailani D and Broze G J Jr., Science. 1991 Aug. 23; 253(5022):909-12; Kravtsov D V et al. Blood. 2009 Jul. 9; 114(2):452-8).
Interestingly, FXI deficiency usually does not lead to spontaneous bleeding, but is associated with increased risk of bleeding with hemostatic challenges, although the severity of bleeding correlates poorly with the plasma level of FXI. Severe FXI deficiency in humans has been reported to have certain protective effects from thrombotic diseases, including ischemic stroke and deep venous thrombosis (DVT) (Salomon O et al, Thromb Haemost. 2011 February; 105(2):269-73; Salomon O et al, Blood. 2008 Apr. 15; 111(8):4113-7). Yet, a high level of FXI has been associated with thrombotic events and has been reported to confer higher risk for DVT, myocardial infarction (MI), and stroke (Meijers J C et al, N Engl J Med. 2000 Mar 9; 342(10):696-701).
Taken together, previous studies suggest that FXI has a minor supporting role in maintaining hemostasis but is a crucial contributor to the pathogenesis of thrombosis, thereby rendering FXI a promising target for antithrombotic therapy. This is so because, although thrombosis and hemostasis are not identical molecular processes, they are similar enough that currently used antithrombotic drugs inadvertently target both. Presently available antithrombotic drugs either target the building blocks of thrombi (fibrin and platelets) or inhibit molecules (coagulation factors) and cells (platelets) from participating in the thrombus-forming process. Antiplatelet, profibrinolytic and anticoagulant agents have been the mainstay for the treatment and prevention of thromboembolic diseases for decades and are among the most commonly prescribed drugs in clinical practice. Yet, most of these agents can completely block both thrombosis and hemostasis when administered in effective doses.
So far, one of the few examples for an anti-FXI antibody exhibiting therapeutic potential is murine antibody 1A6 (also named aXIMab) as published by Tucker et al. (Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI. Erik I. Tucker, Ulla M. Marzec, Tara C. White, Sawan Hurst, Sandra Rugonyi, Owen J. T. McCarty, David Gailani, András Gruber, and Stephen R. Hanson. Blood. 2009 Jan. 22; 113(4):936-944). Antibody 1A6 is also disclosed in patent application WO 2009/067660 A2, also published as U.S. Pat. No. 9,125,895, which is incorporated herein by reference in its entirety. However, as antibody 1A6 is a murine antibody, it is unsuitable for human therapies especially for chronic applications as such as antithrombotic therapy. One method to convert a murine antibody into an acceptable therapeutic antibody is so-called humanization. Standard techniques are available to the person skilled in the art such as those described in O'Brien and Jones, Humanising Antibodies by CDR Grafting, Chapter 40; Antibody Engineering, Part of the series Springer Lab Manuals pp 567-590; R. Kontermann et al. (eds.), Antibody Engineering; Springer-Verlag Berlin Heidelberg 2001 and in Hwang, Almagro, Buss, Tan, and Foote (2005) Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods, May; 36(1):35-42 and in the references therein. For further reduction of the inherent immunogenicity potential of humanized antibodies, further sequence optimization and germlining is required.
When Applicants applied these standard methods to humanize and optimize parenteral antibody 1A6, some of the resulting antibodies exhibited binding activity in a biochemical assay comparable to the parenteral 1A6, however, showed a significant loss of activity in plasma based assays and were inadequate in in vivo models of coagulation. Thus far, there is no explanation readily available why a comparable biochemical profile of the parental murine antibody 1A6 and humanized variants does not translate into efficient anti-thrombotic activity. Surprisingly, by introduction of further sequence alterations a humanized variant of parental murine antibody 1A6 has been generated (antibody TPP-3583) which display both a comparable biochemical profile and anti-thrombotic efficacy in vivo.
With the antibodies of this invention, therapeutic molecules have been generated which have a reduced immunogenicity risk and effectively block thrombosis without debilitating hemostasis, thereby making antithrombotic therapy safer, and thus broadening the range of clinical indications and scenarios in which antithrombotic therapy can be applied.